PROTEOLIPOSOMES
PRESENTED BY GROUP # THREE

INTRODUCTION
•Proteoliposomes are the vesicle systems that mimic lipid
membranes ( lipposomes) to which a protein has been
incorporated or instered.
•During last decade , these systems have gained
prominence as tools for biological studies on lipid –
protein interactions as well as for their biotecnological
Applications.

PRINCIPLE OF FORMATION
• The mechanism of the formation of proteoliposomes has long been
surprisingly not well defined.
• The membrane proteins which are solubilized in Detergent has the
ability to integrate into phospholipid bilayers ( Liposomes) when
detergent is removed by dilution or dialysis.
• The protein, solublized in Detergent is mixed with the prepared
Liposomes. The suspension is diluted with the buffer. The protein
leaves the detergent and proteins becomes incorporated in the
phospholipid bilayer.

PREPARATION OF PROTEOLIPOSOMES
STEP BY STEP PROCEDURE
The new reconstitution Strategy proceeds in four steps..
,
1. Preparation of large , homogeneous, and unilamellar
Liposomes.
2. Addition of detergent to the preformed Liposomes.
3. Addition of Solublized protein.
4. Detergent removal and characterization of the
reconstituted products.

LIPOSOMES ; THE PARENT MOLECULE
• Liposomes are self assembled closesd spherical
structures composed of one or more concentric lipid
bilayers and
• They range in size from 20 nm to many microns.
• They are amphophilic molecules.
• They may be unilamellar or multilamellar.

HISTORY OF LIPOSOMES
• They are first discover by A.D. Bangham in 1965.
• Genesis period ( 1968-75 ): The physical and chemical
characteristics of Liposomes have been studied.
• Middle age ( 1975 -85): During this period , the advantages,
stability and interaction characteristics, physical and chemical
properties of liposomes , interactions with cell studied.
• Modern era ( after 1985) : Today liposomes are used in
different fields , such as biophysics, mathematics,
biochemistry, theoretical physics and biology.

METHODS TO PREPARE LIPOSOMES
The first stage in the reconstitution strategy is to prepare unilamellar
and homogeneous preformed Liposomes.
One of the method to achieve this reverse –phase evaporation method.
A typical preparation contains 50 mg of phospholipids , usually
Solublized in chloroform and dried under high vaccum.
Other methods are;
Sonication
Thin film hydration method followed by extrusion.

ADDITION OF DETERGENT /
SOLUBILIZATION
• Liposomes prepared are diluted with detergent by adding increasing amount
of detergent to aliquoted Liposomes suspension.
• Gradual Solubilization of detergent saturated Liposomes Into small lipid –
detergent micelles.
• At the end of Solubilization process, all Liposomes are Solublized and
solution becomes transparent.

ADDITION OF PROTEINS
•After the various detergent –phosphilipid mixtures have been
equilibrated , the selected membrane protein is added as a
monodisperse detergent protein solution.
•Protein are incubated for 1 h ( varies according to detergent)
with the lipid detergent mixture before detergent removal.

DETERGENT REMOVAL
• The last step of reconstitution is related to the removal of detergent from the
equilibrated lipid- detergent-ptotein mictures.
• Although there are various method of detergent removal , adsorption on Biobeads
SM2 has been demonstrated to be the most efficient removal of all kind of
detergent.
• Washed Biobeads are added directly to each lipid-ptotein – detergent solution.
• The solution is incubated with Biobeads several times to remove all of the
detergent.

SUMMARY

APPLICATIONS OF PROTEOLIPOSOMES

TRANSPORT ACROSS MEMBRANE
•Proteoliposomes are widely used to study the function of
membrane proteins.
•Reconstitution of membrane bound enzymes and transport
Proteins with the artificial phospholipid bilayer (liposomes) is
one of the most useful techniques to study the functional
aspects of these proteins.

ROLE OF LIPOSOMES AS IMMUNOGENS
•Liposomes can be prepared mimicking pathogens.
•Upon vaccination the immune system primarily recognizes
and process them as pathogens.
•Liposomes can encapsulate multiple copies of structurally
epitopes for a complete set of immune responses.
•Proteoliposomes provides an antigenic depot along with the
transport of adjuvants which are required for immunity.

ROLE IN NANOTECHNOLOGY
• Proteoliposomes are one of the first nanotechnologies to benefit patients with
cancer or infectious diseases.
• Liposomal anthracyline formulations have demonstrated to have a great impact in
oncology.
• Deliver highly toxic anti cancer agent in patient with breast carcinoma metastasis.
• Long circulating proteoliposomes, coated with polyethylene glycol , can access
the angiogenic Vessels , and can effectively deliver the drug as compared to free
drug.
• Attaching tumor- specific targeting ligands such as monoclonal antibodies to the
Liposomes surface results in the accumulation of drug at the tumor site.

TRANSPORTERS OF XENOBIOTICS
•Proteoliposomes represent a suitable and up to date tool for
studying membrane transporters which physiologically
mediate absorption, excretion, trafficking and reabsorption of
nutrients and metabolites.
•Proteoliposomes have recently been used for studying the
interaction of some plasma membrane and mitochondrial
transporters with toxic compounds, such as mercurials, H2O2
and some drugs.

LIMITATIONS
1.Liposomes delivered intervaenously can destroyed
by mononuclear phagocyte system.
2.Interaction between Liposomes and lipoproteins in
the blood, which may cause the Liposomes to leak.
3.Proteoliposomes Reconstitution by mechanical
means drastically limited because of local probe
heating